scholarly journals Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis

2002 ◽  
Vol 367 (1) ◽  
pp. 107-111 ◽  
Author(s):  
Satoshi TAMAHARA ◽  
Mutsumi INABA ◽  
Kota SATO ◽  
Naoaki MATSUKI ◽  
Yoshiaki HIKASA ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Glutamate Transporters ◽  
Functional Expression ◽  
Glutamate Transport ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Regulatory Pathways ◽  
Oligomer Formation ◽  
Redox Sensing

A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na+-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg2+ and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters.

scholarly journals Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36

1997 ◽  
Vol 328 (2) ◽  
pp. 635-642 ◽  
Author(s):  
Paola GRUARIN ◽  
Roberto SITIA ◽  
Massimo ALESSIO
Keyword(s):  
Scavenger Receptor ◽  
Extracellular Domain ◽  
Low Density Lipoproteins ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Mobility Shift ◽  
Disulphide Bonds ◽  
Gel Mobility Shift ◽  
Triton X

In monocytes/macrophages, CD36 is thought to have a role as a scavenger receptor, mediating the phagocytosis of apoptotic cells and the endocytic uptake of oxidized low-density lipoproteins and fatty acids. The proposed topology of CD36 predicts that, of ten cysteine residues, six lie in the extracellular domain, whereas four are equally distributed in the two short terminal tails flanking the N-terminal and C-terminal hydrophobic stretches. Here we investigate the formation of intrachain disulphide bonds, on the basis of the assumption that the cysteine residues present in the luminal domains are generally oxidized, whereas those in the cytosol are reduced. As revealed by gel mobility-shift assays, disulphide bonds are present in the extracellular domain of the CD36 molecule. The formation of these bonds is required for the transport of CD36 from endoplasmic reticulum to Golgi. Furthermore reactive thiol groups are present in the CD36 sequence, which upon lysis form an intrachain extra loop as an artifact. This disulphide bond is not formed in either (1) truncated CD36 lacking the two C-terminal cysteine residues or (2) Triton X-100-insoluble wild-type CD36 molecules, suggesting that, in this fraction, the C-terminal thiol groups are modified.

scholarly journals Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

2005 ◽  
Vol 386 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sandra MÜLLER ◽  
Manuela SCHÖTTLER ◽  
Sylvia SCHÖN ◽  
Christian PRANTE ◽  
Thomas BRINKMANN ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals Plant Hormone Metabolome and Transcriptome Analysis of Dwarf and Wild-type Banana

2021 ◽  
Author(s):  
Biao Deng ◽  
Xuan Wang ◽  
Xing Long ◽  
Ren Fang ◽  
Shuangyun Zhou ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Transcriptome Analysis ◽  
Regulatory Mechanism ◽  
Feedback Regulation ◽  
Wild Type ◽  
Hormone Levels ◽  
Transporter Protein ◽  
Repressor Proteins ◽  
Differential Gene ◽  
The Relationship

AbstractGibberellin (GA), auxin (IAA) and brassinosteroid (BR) are indispensable in the process of plant growth and development. Currently, research on the regulatory mechanism of phytohormones in banana dwarfism is mainly focused on GA, and few studies are focused on IAA and BR. In this study, we measured the contents of endogenous GA, IAA and BR and compared the transcriptomes of wild-type Williams banana and its dwarf mutant across five successive growth periods. We investigated the relationship between hormones and banana dwarfism and explored differential gene expression through transcriptome analysis, thus revealing the possible metabolic regulatory mechanism. We inferred a complex regulatory network of banana dwarfing. In terms of endogenous hormone levels, GA and IAA had significant effects on banana dwarfing, while BR had little effect. The key gene in GA biosynthesis of is GA2ox, and the key genes in IAA biosynthesis are TDC and YUCCA. The differential expression of these genes might be the main factor affecting hormone levels and plant height. In terms of hormone signal transduction, DELLA and AUX/IAA repressor proteins were the core regulators of GA and IAA, respectively. They inhibited the process of signal transduction and had feedback regulation on hormone levels. Finally, the transporter protein PIN, AUX1/LAX protein family and ABCB subfamily played supplementary roles in the transport of IAA. These results provide new insights into GA and IAA regulation of banana growth and a reliable foundation for the improvement of dwarf varieties.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Binding Component of Clostridium perfringens Iota-Toxin Induces Endocytosis in Vero Cells

2002 ◽  
Vol 70 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Masahiro Nagahama ◽  
Koichi Nagayasu ◽  
Keiko Kobayashi ◽  
Jun Sakurai
Keyword(s):  
Clostridium Perfringens ◽  
Vero Cells ◽  
Binary Toxin ◽  
Wild Type ◽  
Oligomer Formation ◽  
Clostridium Perfringens Iota Toxin ◽  
Binding Component ◽  
Pronase Treatment

ABSTRACT Clostridium perfringens iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K+ release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K+ release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.

scholarly journals Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling

2005 ◽  
Vol 25 (15) ◽  
pp. 6722-6733 ◽  
Author(s):  
Sandrine Roy ◽  
Sarah Plowman ◽  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Lipid Rafts ◽  
Lipid Bilayer ◽  
Spatial Organization ◽  
Cysteine Residues ◽  
Wild Type ◽  
Membrane Affinity ◽  
Lateral Segregation

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

scholarly journals Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

2004 ◽  
Vol 15 (8) ◽  
pp. 3891-3902 ◽  
Author(s):  
Ramila S. Patel-King ◽  
Oksana Gorbatyuk ◽  
Sachiko Takebe ◽  
Stephen M. King
Keyword(s):  
Regulatory Mechanism ◽  
Signal Transduction Pathway ◽  
Nucleoside Diphosphate Kinase ◽  
Terminal Segment ◽  
Transduction Pathway ◽  
Wild Type ◽  
Repetitive Region ◽  
Independent Manner ◽  
Calmodulin Binding ◽  
Radial Spokes

The radial spokes are required for Ca2+-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca2+-independent manner, whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca2+. This Ca2+-responsive enzyme, which accounts for ∼45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca2+-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.

Functional and structural comparisons of cysteine residues in the Val108 wild type and Met108 variant of human soluble catechol O-methyltransferase

2005 ◽  
Vol 152 (2-3) ◽  
pp. 151-163 ◽  
Author(s):  
Yan Li ◽  
Xiaofeng Yang ◽  
Minsun Chang ◽  
James D. Yager ◽  
Richard B. van Breemen ◽  
...  
Keyword(s):  
Cysteine Residues ◽  
Wild Type
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